Structure And Function Of Peptides Obtained From Odorous Frogs

Objectives:Odorous frog skins display considerable bioactivity,including defense,respiration,excretion,body color adjustment,ect.The skin secretions contain various peptides which have specific biological activity,including wound-healing-promoted activity,antimicrobial activity,antioxidant activity,immunomodulatory activity,ect The study on odorous frog skin secretions will help in the development of novel medicine agents and in our understanding of the biological functions of amphibians.Methods:By the use of Sephadex G-75 gel filtration column and RP-HPLC to purify the skin secretions,then collect the antioxidant samples that against ABTS+free radicals to determine the peptide primary structure;Cloning of cDNA encoding the antioxidant peptide;antioxidant peptide synthesis;to test samples antioxidant activity(ABTS+,DPPH,Fe3+ and NO free radicals scavenging activity),antimicrobial activity,accelerate wound-healing activity,immunomodulatory activity(TNF-a and TGF-?1),hemolytic activity and acute toxicity assay;using HE and immunohistochemistry for histological analysis.Results:(1)We identified a novel cathelicidin from amphibian skin secretion,named as cathelicidin-OA1(OA:odorrana andersonii).Produced by posttranslational processing of a 198-residue prepropeptide,cathelicidin-OA1 presented an amino acid sequence of 'IGRDPTWSHLAASCLKCIFDDLPKTHN'and a molecular mass of 3037.5 Da,with 2 Da difference from mass spectrometry observed molecular mass,which indicated the formation of an intramolecular disulfide bridge.Unlike other cathelicidin,cathelicidin-OA1 peptide showed no antimicrobial activity,hemolytic activity and acute toxicity,but did exhibit antioxidant activity against ABTS+ and DPPH free radicals.Furthermore,cathelicidin-OA1 also exhibited promoted wound-healing activity.In vitro,cathelicidin-OA1 promoted wound-healing activity against human keratinocytes(HaCaT)and skin fibroblasts(HSF)in both time-and dose-dependent manners.And in vivo,cathelicidin-OA1 also promoted wound-healing in a mouse model with full-thickness skin wounds,accelerating re-epithelialization and granulation tissue formation by enhancing the recruitment of macrophages to the wound site with relations of TNF-a and TGF-1?.(2)We identified a novel gene-encoded antioxidant peptide from odorrana andersonii,named as OA-VI12(OA:species abbreviation VI:two initial amino acids 12:peptide length).OA-VI12 was produced by the post-translational processing of a 59-residue prepropeptides and the amino acid sequence of the peptide was'VIPFLACRPLGL',with a molecular mass of 1298.65 Da and no observed post-transcriptional modifications.For the peptide OA-VI12,the C7 amino acid was responsible for the scavenging ABTS+ and Fe3+ free radicals,the F4,C7,and P9 amino acids were crucial for the scavenging DPPH free radicals,and the P9 amino acid was responsible for the scavenging NO free radicals.(3)We identified another gene-encoded antioxidant peptide from amphibian species,odorrana margaretae,named as OM-GF17(OA:species abbreviation GF:two initial amino acids 17:peptide length).Produced by the post-translational processing of a 61-residue prepropeptide,OM-GF17 presented an amino acid sequence of 'GFFKWHPRCGEEHSMWT' and a molecular mass of 2135.7 Da,with no post-transcriptional modifications.Functional analysis,OM-GF17 showed antioxidant activity against ABTS+,DPPH,Fe3+ and NO free radicals.For the peptide of OM-GF17,we determined that the F2/3,W5/16,P7,C9?and M15 amino acid residues were jointly responsible for the scavenging ABTS+ free radicals,the F2/3,M15,and W16 amino acid residues were crucial for the scavenging DPPH free radicals,the C9 amino acid residue was responsible for the scavenging Fe3+ free radicals,and the F2/3 and M15 amino acid residues were essential for the scavenging NO free radicals.Conclusions:(1)We identified a novel antioxidant peptide,named as cathelicidin-OAl,presented an amino acid sequence of'IGRDPTWSHLAASCLKCIFDDLPKTHN',with an intramolecular disulfide bridge formed between two cysteines.Unlike other cathelicidin,cathelicidin-OA1(IGRDPTWSHLAASCLKCIFDDLPKTHN)exhibited no direct microbe-killing,hemolytic activity and acute toxicity,but did exhibit antioxidant activity and promoted wound-healing potency in vitro and in vivo.(2)OA-VI12 showed that it could scavenge ABTS+,DPPH,Fe3+,and NO free radicals.And the C7 amino acid was responsible for the scavenging ABTS+ and Fe3+free radicals,the F4,C7,and P9 amino acids were crucial for the scavenging DPPH free radicals,and the P9 amino acid was responsible for the scavenging NO free radicals.(3)For another peptide OM-GF17 showed that it could scavenge ABTS+,DPPH,Fe3+,and NO free radicals.Furthermore,for the peptide of OM-GF17,we determined that the F2/3,W5/16,P7,C9,and M15 amino acid residues were jointly responsible for the scavenging ABTS+ free radicals,the F2/3,M15,and W16 amino acid residues were crucial for the scavenging DPPH free radicals,the C9 amino acid residue was responsible for the scavenging Fe3+ free radicals,and the F2/3 and M15 amino acid residues were essential for the scavenging NO free radicals.